*/Disease Information (sort of like the Intro to your Mini Research Write up): Giardiasis is a common intestinal infection caused by the exposure to the organism Giardia lamblia [1]. Exposure to contaminated water, food, or direct contact with the organism can lead to the disease [1]. Giardia is one of the most common intestinal parasites in the world making Giardiasis a very common infection. Cases are becoming increasingly common in the United States. A well-known cause of Giardiasis is through drinking unfiltered water that may be contaminated with human or animal waste [1]. This has brought the infectious disease to be highly associated with nicknames “travelers diarrhea” and “beaver fever”. One of the main concerns is that children can easily spread the infection in a daycare environment. The increased contact between children and each other’s feces makes it a likely spreading ground [1]. When the inactive forms of Giardia (Cysts) are ingested, the acid within the stomach releases the parasite from the cyst. From there the parasite latches to the lining of the small intestine and reproduces. Cysts are then passed on through the fecal matter of the host [1]. Giardiasis is non fatal and a large number of those infected never display symptoms. When symptoms are displayed they often involve watery and aggressive diarrhea other symptoms include stomach pain, nausea, and vomiting. The disease can often be allowed to run its course or treated with a multitude of antibiotics.
[1] Carol A. Turkington, Giardiasis, The Gale Encyclopedia of Medicine, 2011, vol. 3, (4th ed.), 1889-1892 Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) https://www.ncbi.nlm.nih.gov/gene/5701142
Essentiality of this protein: The Carbamate Kinase is essential to the function of Giardia Lamblia as is is part of a pathway that contributes to energy production. Carbamate Kinase catalyzes the reversible conversion of carbamoyl phosphate and ADP to ATP, ammonia and CO2
Is it a monomer or multimer as biological unit? Multimer as a biological unit
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 34.220
Molar Extinction coefficient of your protein at 280 nm wavelength: Ext. coefficient 28420 Abs 0.1% (=1 g/l) 0.830, assuming all Cys residues are reduced
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
*NCBI Gene # or RefSeq#:
GL50803_16453
*Protein ID (NP or XP #) or Wolbachia#:
NW_002477112.1
*Organism (including strain): Giardia Lamblia
Etiologic Risk Group (see link below):
Group 1
http://www.bacterio.net/-hazard.html#group1
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Giardiasis is a common intestinal infection caused by the exposure to the organism Giardia lamblia [1]. Exposure to contaminated water, food, or direct contact with the organism can lead to the disease [1]. Giardia is one of the most common intestinal parasites in the world making Giardiasis a very common infection. Cases are becoming increasingly common in the United States. A well-known cause of Giardiasis is through drinking unfiltered water that may be contaminated with human or animal waste [1]. This has brought the infectious disease to be highly associated with nicknames “travelers diarrhea” and “beaver fever”. One of the main concerns is that children can easily spread the infection in a daycare environment. The increased contact between children and each other’s feces makes it a likely spreading ground [1]. When the inactive forms of Giardia (Cysts) are ingested, the acid within the stomach releases the parasite from the cyst. From there the parasite latches to the lining of the small intestine and reproduces. Cysts are then passed on through the fecal matter of the host [1]. Giardiasis is non fatal and a large number of those infected never display symptoms. When symptoms are displayed they often involve watery and aggressive diarrhea other symptoms include stomach pain, nausea, and vomiting. The disease can often be allowed to run its course or treated with a multitude of antibiotics.
[1] Carol A. Turkington, Giardiasis, The Gale Encyclopedia of Medicine, 2011, vol. 3, (4th ed.), 1889-1892
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
https://www.ncbi.nlm.nih.gov/gene/5701142
Essentiality of this protein:
The Carbamate Kinase is essential to the function of Giardia Lamblia as is is part of a pathway that contributes to energy production.
Carbamate Kinase catalyzes the reversible conversion of carbamoyl phosphate and ADP to ATP, ammonia and CO2
Is it a monomer or multimer as biological unit? Multimer as a biological unit
Complex of proteins?: yes
http://www.rcsb.org/structure/4OLC
Druggable Target :
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3565245/ (inhibition in Giardia Lamblia)
*EC#: 2.7.2.2
Link to BRENDA EC# page:
https://www.brenda-enzymes.org/enzyme.php?ecno=2.7.2.2
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
ADP + carbamoyl phosphate
ATP + carbamate
ATP + NH3 + CO2
-- link to Sigma (or other company) page for assay (see Sigma links below)
https://www.sigmaaldrich.com/catalog/product/supelco/46856u?lang=en®ion=US
https://www.sigmaaldrich.com/catalog/product/sigma/flaas?lang=en®ion=US
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 4OLC
Current Inhibitors: Disulfarim
https://www.ncbi.nlm.nih.gov/pubmed/24558036
Expression Information (has it been expressed in bacterial cells):
Yes, Previously Expressed in E.Coli Cells (BL21) DE3)Star as a TEV protease-cleavable, His-tagged, maltose binding protein fusion protein
Purification Method:
possible method: https://www.sciencedirect.com/science/article/pii/0926656964900100
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSAGKTVVIA LGGNAMLQAK EKGDYDTQRK NVEIAASEIY KIHKAGYKVV LTSGNAPQVG AIKLQNQAAA GVSPEMPLHV CGAMSQGFIG YMMSQAMDNV FCANNEPANC VTCVTQTLVD PKDQAFTNPT KPRWRFYTEQ EAKDLMAANP GKILREDAWP AGWRVVVPSP RPLEIVEYGV IKTLIDNNVL VICTNGGGIP CKRENKVISG VDAVIDKDLA TSLLAKTLNS DYLMILTDVL NACINYKKPD ERKLEEIKLS EILALEKDGH FAAGSMGPKV RAAIEFTQAT GKMSIITSLS TAVDALNGKC GTRIIKD
*length of your protein in Amino Acids: 317
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 34.220
Molar Extinction coefficient of your protein at 280 nm wavelength: Ext. coefficient 28420 Abs 0.1% (=1 g/l) 0.830, assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
GGACCTGCTAACCCTTGCTGTGGTGTCTACAGGAGTCACTGCGGGGGCGCTTTCCTCCATAACACTTGTA
GAGTAAAGAATGATCTAAAGTTTTTGATCGTTAAAAGCCTCAGACGCCCTTGAGGCGGAAAATAAATTCC
AGATGTCGGCAGGGAAAACGGTTGTGATTGCGCTGGGCGGCAACGCTATGCTTCAGGCCAAGGAGAAGGG
GGACTATGACACTCAACGTAAGAATGTTGAGATTGCGGCCTCGGAGATTTATAAAATCCATAAGGCTGGC
TATAAGGTTGTTCTCACAAGTGGTAACGGGCCGCAGGTGGGTGCCATAAAACTCCAAAACCAGGCTGCAG
CAGGTGTTTCTCCAGAAATGCCCCTCCACGTCTGTGGTGCAATGTCCCAGGGATTCATCGGCTACATGAT
GTCTCAGGCGATGGACAACGTTTTTTGCGCGAATAACGAGCCAGCAAACTGTGTTACCTGTGTCACTCAG
ACATTAGTCGATCCGAAGGATCAGGCTTTCACAAATCCCACAAAGCCCGTTGGGAGGTTCTACACCGAGC
AGGAGGCGAAGGACCTCATGGCTGCAAACCCGGGAAAAATCCTCCGCGAGGATGCTGGCCGCGGCTGGCG
TGTTGTCGTCCCATCTCCTCGTCCTCTTGAGATCGTGGAGTATGGTGTCATCAAGACCCTCATTGACAAC
AACGTTCTTGTCATTTGTACGAATGGGGGAGGAATCCCTTGCAAGCGCGAGAACAAGGTCATCAGTGGCG
TGGATGCTGTCATCGACAAGGACCTTGCAACCTCTCTTCTAGCTAAAACGCTGAACTCGGACTACCTTAT
GATACTTACGGACGTCCTCAACGCGTGCATCAATTACAAGAAGCCCGATGAGAGAAAGCTCGAGGAGATC
AAGCTTAGCGAGATTCTCGCCCTCGAAAAGGACGGCCACTTTGCTGCCGGATCTATGGGGCCGAAGGTCC
GTGCAGCAATCGAGTTTACACAGGCAACTGGAAAAATGAGCATCATCACCTCCCTGAGCACTGCGGTGGA
TGCGCTCAATGGTAAATGTGGGACCCGCATCATCAAGGATTAACTTTGAAGCGAGGCTGTACGCTCCCAC
TCATATATCAGTGAATTTTCCAGCTGCTCTACCTTCGTCATTAGCCGCATAATCTCGTCCTCGTATGGCT
TGCCTGCCCTGTTGAGCCGAAGAGCAGTATTCAGCTGTTCGATCTCT
*GC% Content for gene: 51.4%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.